Fluorescence tools for studying DNA-protein interactions with application in the investigation of Human Maturation of Okazaki Fragments
KAUST DepartmentBiological and Environmental Science and Engineering (BESE) Division
Embargo End Date2022-11-30
Permanent link to this recordhttp://hdl.handle.net/10754/673847
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Access RestrictionsAt the time of archiving, the student author of this dissertation opted to temporarily restrict access to it. The full text of this dissertation will become available to the public after the expiration of the embargo on 2022-11-30.
AbstractFluorescence-based assays have gained an ever-increasing popularity in life sciences. One of these rapidly emerging techniques is Protein Induced Fluorescence Enhancement (PIFE). Traditional explanations of PIFE focused exclusively on the role of the protein and largely neglected the role of the mediator DNA. In the same time, the existing models of PIFE were denying its exactly opposite effect. In the first part of the current dissertation we focus on a better understanding of PIFE, stimulated by the direct observation of its opposite effect, Induced Fluorescence Enhancement Quenching (PIFQ). This study offered us the leverage for obtaining on-demand fluorescence modulation in cyanine dyes. The following two chapters harvest this control over fluorescence modulation to generate two biotechnology applications: a sensitive potassium sensor with embedded fluorescent transducer, and a simple protocol for the fluorescent detection of His-tagged proteins. In the last part, a variety of fluorescence tools including Förster resonance energy transfer, fluorescence enhancement, and fluorescence quenching are employed for a much more complex task; to demystify the behavior of the human Maturation of Okazaki Fragments (MOF) machinery. First, we reconstituted the human MOF reaction and showed that it behaves considerably different than its well-established yeast homolog. Subsequently, our toolbox of fluorescence-based assays was used to pinpoint the kinetics and dynamics that lead to this unexpected MOF behavior.
CitationRaducanu, V.-S. (2021). Fluorescence tools for studying DNA-protein interactions with application in the investigation of Human Maturation of Okazaki Fragments. KAUST Research Repository. https://doi.org/10.25781/KAUST-0P4ZI